重组人乙型肝炎病毒核糖核酸酶H基因工程菌的优化表达及其单克隆抗体制备
发布时间：2019-03-18 10:24
【摘要】：目的:利用基因工程技术,分别构建含HBV/RNAse H1及H2基因的原核表达载体pGSTag,并转化到大肠杆菌,诱导表达出目的蛋白后,用原核表达产物作为抗原,制备单克隆抗体,并对抗单克隆抗体血清型进行初步分析鉴定,为临床应用奠定基础。 方法:本研究采用摇瓶发酵,将重组质粒pGSTag/HBV/RNAseH1及H2分别转化BL21(DE3)codon plus(?)-RP、HB101、BL21(DE3)PlysS、G1727、DH5α、BL21大肠杆菌。同时用不同的培养基(LB、SOB、SOC、TB、2YT、M9)、不同的初始菌浓度(即OD为0.2,0.4,0.6,0.8,1.0,1.2)、不同的诱导时间(2.0h,4.0h,6.0h)、初始pH(6.0,6.5,7.0,7.5,8.0,8.5)以及不同的IPTG诱导浓度(0.1,0.3,0.6,1.0,2.0,3.0mmol/L),分别表达重组蛋白,并将重组菌与空白菌作生长曲线对比,收集菌体,初步纯化,菌液(0.5ml)经离心洗涤后,进行SDS-PAGE分析,并用FR-200紫外可见分析装置、复日smart view 2001生物电泳图象分析系统分析pGSTag/HBV-RNAseH1及H2蛋白表达量,同时进行Westerblot印迹分析。并以
[Abstract]:The invention aims to construct a prokaryotic expression vector pGSTag containing the HBV/ RNAse H1 and the H2 gene by using the genetic engineering technology, and is transformed into E. coli to induce the expression of the target protein, and the prokaryotic expression product is used as an antigen to prepare the monoclonal antibody. And a preliminary analysis and identification of the monoclonal antibody serotype is carried out to lay a foundation for clinical application. Methods: The recombinant plasmid pGSTag/ HBV/ RNAseH1 and H2 were transformed into BL21 (DE3) coon plus (?)-RP, HB101, BL21 (DE3) PlysS, G1727 and DH5 by shake flask fermentation. BL21. coli. At the same time, different culture media (LB, SOB, SOC, TB, 2YT, M9), different initial bacterial concentrations (i.e., OD of 0.2, 0.4, 0.6, 0.8, 1.0, 1.2), different induction times (2.0 h, 4.0 h, 6.0 h), initial pH (6.0, 6.5, 7.0, 7.5, 8.0, 8.5), and different IPTG-induced concentrations (0.1, 0.3, 0.6, 1.0, 2.0, 3.0 mmol/ L), The recombinant protein was expressed separately, and the recombinant bacteria were compared with the blank fungus as the growth curve. The cells were collected, and the bacterial liquid (0.5 ml) was purified by centrifugation. The SDS-PAGE analysis was carried out. and the expression of the H2 protein is carried out at the same time, Bl
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R392
[Abstract]:The invention aims to construct a prokaryotic expression vector pGSTag containing the HBV/ RNAse H1 and the H2 gene by using the genetic engineering technology, and is transformed into E. coli to induce the expression of the target protein, and the prokaryotic expression product is used as an antigen to prepare the monoclonal antibody. And a preliminary analysis and identification of the monoclonal antibody serotype is carried out to lay a foundation for clinical application. Methods: The recombinant plasmid pGSTag/ HBV/ RNAseH1 and H2 were transformed into BL21 (DE3) coon plus (?)-RP, HB101, BL21 (DE3) PlysS, G1727 and DH5 by shake flask fermentation. BL21. coli. At the same time, different culture media (LB, SOB, SOC, TB, 2YT, M9), different initial bacterial concentrations (i.e., OD of 0.2, 0.4, 0.6, 0.8, 1.0, 1.2), different induction times (2.0 h, 4.0 h, 6.0 h), initial pH (6.0, 6.5, 7.0, 7.5, 8.0, 8.5), and different IPTG-induced concentrations (0.1, 0.3, 0.6, 1.0, 2.0, 3.0 mmol/ L), The recombinant protein was expressed separately, and the recombinant bacteria were compared with the blank fungus as the growth curve. The cells were collected, and the bacterial liquid (0.5 ml) was purified by centrifugation. The SDS-PAGE analysis was carried out. and the expression of the H2 protein is carried out at the same time, Bl
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R392
【参考文献】
相关期刊论文 前3条
1 张惠中,程虹;乙型肝炎病毒核糖核酸酶H基因表达及鉴定[J];第四军医大学学报;2000年03期
2 邱英华,沈益,王玉海,徐贤秀,董雪吟,张洪祖;Cecropin-X发酵过程中工程菌质粒稳定性的研究[J];微生物学报;2004年03期
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